Experiments described in this proposal will explore the role of protein-DNA interactions in promoter function of the long terminal repeats of two retroviruses, Rous sarcoma virus and mouse mammary tumor virus. A correlation will be made between protein binding to sequence elements of the RSV LTR, as detected by the gel mobility shift assay; and the role of those same sequence elements in promoter function. Sequence-specific probes and high-resolution mapping techniques will be used to map protein binding sites, both in vitro and in vivo on integrated proviruses. Special emphasis will be given the use of uv light to "photofootprint" protein-dependent changes in DNA structure that accompany promoter activation. The patterns of these changes will be carefully examined in search for clues for mechanisms of enhancer action. These same procedures will also be applied to the problem of glucocorticoid receptor activation of the MMTV promoter. MMTV LTR fragments will be placed on bovine papilloma virus vectors and then subjected to photofootprint analysis. At the same time large amounts of receptors protein will be made by expressing it in insect cells from a baculovirus vector. The protein will be added to nuclei in an attempt to generate in vitro, the same changes in promoter structure that are seen in vivo. Finally, the hormone response elements of an MMTV virus construction will be replaced with the enhancer sequences of RSV. Patterns of expression of this hybrid will be examined in the mouse to determine the role of the hormone element in mammary gland specific expression.